Protective effect of quercetin on skeletal and neural tube teratogenicity induced by cyclophosphamide in rat fetuses

Cyclophosphamide (CP) is a drug commonly used to treat neoplastic disease and some autoimmune diseases. It is also a well-known and well-studied teratogen causing a variety of birth defects in fetuses of pregnant women treated with the drug. There are many reports that show the adverse effects of CP can be decreased by use of antioxidant drugs. It appears that, quercetin has antioxidant effect. The aim of this study was prevention or decrease of teratogenicity of CP in fetuses of rats by quercetin. This study was performed on 35 pregnant rats divided into six groups. Control group was received normal saline (5 mL kg-1, intraperitoneally) and 2-6 groups received a single dose of CP (15 mg kg-1), a single dose of quercetin (75 or 200 mg kg-1), CP plus quercetin (75 or 200 mg kg-1) intraperitoneally at 9th day of gestation, respectively. Fetuses were collected at 20th day of gestation and after determination of weight and crown rump length were stained by alizarin red – alcian blue method and skeletal system were examined by stereomicroscope. The results showed that the cleft palate, exencephaly, spina bifida and omphalocele incidence were 55.56%, 27.77%, 33.34% and 11.11%, in fetuses of rat that received only CP, respectively. However, it decreased to 16.00%, 16.00%, 16.00% and 8.00% by quercetin (75 mg kg-1) and so to 12.90%, 12.90%, 6.45% and 3.28% by quercetin (200 mg kg-1), respectively. On the basis of results, quercetin significantly can decrease teratogenicity induced by CP.


Introduction
Some chemical agents and drugs can induce teratogenic effects and abortion. 1 Developmental defects are major health problems as in the USA 3.00 to 5.00% of fetuses have congenital abnormality. 2 It is estimated that 7.00 to 10.00% of human anatomic anomalies result from the disruptive actions of drugs, viruses, and other environmental factors. 3 De Sanntis et al. also estimated that defects attributable to drug therapy represent about 1.00% of congenital defects of known etiology. 4 Cyclophosphamide (CP) is a drug commonly used to treat neoplastic disease and some autoimmune diseases. It is also a well-known and well-studied teratogen causing a variety of birth defects in the fetuses of pregnant women treated with the drug. 5 Cyclophosphamide, a nitrogen mustard compound, is a member of the group of cytostatic alkylating agents and has several toxic effects including hemorrhagic cystitis. 6 Metabolites of CP, especially acrolein modulates its toxic effects. 3 In order to cause teratogenesis, CP must be bioactivated through a process involving oxidase enzymes that convert it into its active metabolites, phosphoramide mustard and acrolein. 7 Phosphoramide mustard acts to inhibit DNA synthesis and causes cross-links in the existing DNA resulting in cell death, and acrolein is thought to be responsible for some of the side effects of CP chemotherapy, such as cystitis. 3 Although the mechanism of teratogenesis is still debated, it is believed that generation of reactive oxygen species (ROS) through these metabolites plays a role in CP-induced malformations. 7,8 Previous studies in rodents have shown that exposure to CP during organogenesis caused an embryonic and fetal resorption, growth retardation, or multiple anomalies, including exencephaly and limb and skeletal defects. 9 Free radicals or ROS are by-products of the breakdown of many drugs. 10 The exposure of the embryo or fetus to ROS is normally carefully timed so that exposure occurs when antioxidant levels are also high, potentially decreasing the duration of the ROS signal and enabling the cell to repair damage to its DNA. 10 However, exposure to excessive levels of ROS without sufficient antioxidant presence can cause brain and spinal cord defects, embryonic death, or skeletal malformations. 10 Oxidative stress can be prevented by antioxidants known to be effective in vitro for protection against conditions associated with oxidative damage through radical scavenging. 11 Antioxidant agents such as squalene, 12 melatonin, 13 glutamine, 14 and S-allylcysteine 15 have protective actions against CP-induced toxicity. Thus, a combination of the drug delivered together with a potent antioxidant may be appropriate to reduce the toxic side effects of CP.
On the other hands, quercetin, commonly named sophretin and meletin, is a herbal flavonoid found in abundance in apple, onion, tea, green tea leaf, strawberries, broccoli and other plants. Quercetin also has antiinflammatory, anti-bacterial and antioxidant and is used in the prevention of cancer and cardiovascular disease. 16 Quercetin is a powerful antioxidant and free radical scavenger, more powerful than other antioxidants such as vitamin E, vitamin C, which prevents lipid peroxidation. 17 Quercetin supplementation to the diet of pregnant mice reduces fetal malformations caused by methylnitrosourea such as fingers and toes abnormalities. It induces fetal abnormalities via oxidative stress and free radicals. 18 Cyclophosphamide can be teratogenic via oxidative stress. So far, the effects of quercetin have not been studied on CP-induced skeletal malformations in rat fetuses. In the present study, the prophylactic effect of quercetin on CPinduced neural tube defects and skeletal malformations in rat fetuses was evaluated.

Materials and Methods
Male and female healthy Wistar rats, 3 to 4 months of age, weighting 200 to 220 g were purchased (Jundishapour Laboratory Animal Center, Ahvaz, Iran) and housed individually (males) or in 10 per poly-carbonate cage (females) for a 2-week acclimation period. Rats were fed ad libitum by standard laboratory pellet (Pars Khurake-Dam, Tehran, Iran) and tap water. A 12 hr light: 12 hr dark was exercised. Room temperature was at 23 ± 2 ˚C with a relative humidity of 45.00 to 55.00%. This experimental study was conducted in Department of Basic Sciences of Faculty of Veterinary Medicine of Shahid Chamran University (Ahvaz, Iran). The animal care was provided under the supervision of a qualified veterinarian.
Females were mated overnight with males. Pregnancy was ascertained the next morning by presence of a vaginal plug, and this time was designated as gestational day (GD) 0. Ten rats were used in each group (total 60 rats) but 35 rats were harvested as pregnant. Thus, animals in each group were not equal. Pregnant rats (n = 35) were randomly divided into six groups (28 pregnant rats in treatment groups, seven pregnant rats in control group) and treated as follows: Group 1 (control group): Normal saline (5 mL kg -1 ) was administrated to pregnant rats for inducing similar condition (injection and handling) to other groups.
Cyclophosphamide (Baxter Oncology GmbH, Halle, Germany) and quercetin (Sigma-Aldrich, St. Louis, USA) were purchased. The animals were euthanized by diethyl ether and cervical dislocation at 20 th day of gestation. Following laparotomy, the uterus was exteriorized and the number and location of fetuses and resorption were noted, then their weight and crown -rump length (CRL) were measured. Individual fetuses were examined carefully for external anomalies then were stained in a mixture of 0.14% alcian blue and 0.12% alizarin red S in ethanol and glacial acetic acid. Fetuses were then macerated in 2.00% KOH, cleared and hardened in 1:1 glycerin and distilled water, and stored in pure glycerin 21 and investigated by stereomicroscope (Model SMZ200; Nikon, Tokyo, Japan) for skeletal malformations. The incidence of skeletal malformations was determined and compared between groups.
Statistical significance between groups was determined using SPSS (Version 16; SPSS Inc., Chicago, USA) and compared by one way analysis of variance (ANOVA) followed by least significant difference (LSD) post hoc comparison. The minimum level of significance was p < 0.05.

Results
No maternal deaths were observed throughout the course of this study. Likewise, the dose of CP used in this investigation was well tolerated by the dams.

A B C
Open eye and omphalocele, delay ossification in forelimb and several anomalies in sternum were observed (Figs. 3, 4 and 5). Teratogenicity in groups that received CP was similar to groups that received CP plus quercetin, but incidence was lower (Table 1). These anomalies were not observed in animals treated with quercetin. Mean weight and CRL (p < 0.001) were significantly decreased in the group which received only CP. The means weight and length in groups that received CP plus quercetin was greater than the group received only CP except with CP plus quercetin (200 mg kg -1 ), ( Table 2). The mean weight and CRL in the group that received quercetin were significantly decreased in comparison with control group (p < 0.001).

Discussion
Since there are not data available on quercetin on the teratogenicity of CP in rat fetuses. In the present study, for first time, the effect of quercetin on teratogenicity of CP in rat fetuses was evaluated. We demonstrated CP, at dose of 15 mg kg -1 , decreased weight and length and produced cleft palate (55.56%), exencephaly (27.77%), spina bifida (33.34%) and omphalocele (11.11%) among all fetuses. The results presented here show that quercetin administration during the gestational period has a partial protective effect on CP-induced terato-genesis (decreasing the frequencies of exencephaly, cleft palate, spina bifida and omphalocele). In the present study, quercetin reduced the frequency of incidence of neural tube and skeletal fetal defects. Quercetin with dose of 200 mg kg -1 was more effective on decreasing the incidence of neural tube and skeletal fetal defects than 75 mg kg -1 , but it is not significant.
It is well known that CP causes fetal defects in diverse species of animals including mice, rats, hamsters, and rabbits as well as humans. 22 In the present study, a single intraperitoneal administration of CP (15 mg kg -1 ) on GD9 caused significant growth retardation and morpho-logical alterations in rat fetuses.
Gibson and Becker reported CP-induced teratogenicity in mice. They used intraperitoneal CP at dose 5 to 20 mg kg -1 in mice in one of 9 th to 14 th day of gestation. They observed the CP could produce teratogenicity in 67.30% of fetuses with 20 mg kg -1 . 23 They determined fetal defects similar with our study including cleft palate, exencephaly. These anomalies were decreased by 75 mg kg -1 and 200 mg kg -1 quercetin, respectively. They also determined fetal weights and crown rump lengths similar with our study reduced significantly by CP. In present study fetal weights and crown rump lengths were increased by 75 mg kg -1 and 200 mg kg -1 quercetin, respectively in comparison with CP.
Sloth and Hales evaluated effect of mesna on CPinduced teratogenicity. They used CP at dose 10 and 15 mg kg -1 in rats in 13 th day of gestation. They observed the CP could produce teratogenicity in 50.00% and 100% of fetuses with 10 and 15 mg kg -1 , respectively. 19 They determined fetal defects similar with our study including cleft palate, exencephaly, open eye and limb defects. These anomalies were decreased by 75 mg kg -1 and 200 mg kg -1 quercetin, respectively.
Logsdon et al. reported CP at dose 20 mg kg -1 in mice in on 10 th day of gestation could produce teratogenicity and exposure of a developing mammal to moderate doses of green tea as antioxidant can modulate the effects of exposure to CP during development, possibly by affecting biotransformation, while a higher GTE dose tended to exacerbate the developmental toxicity of CP. 24 They determined fetal defects similar with our study including fused or dumbbell-shaped vertebral centra and limb defects. These anomalies were decreased by 75 mg kg -1 and Oxidative stress in any tissue results from an imbalance between the production of ROS such as superoxide anion, hydrogen peroxide, and the hydroxyl ion. A number of teratogens including anti-neoplastic agents have been shown to initiate potentially embryopathic oxidative stress. 26 Cyclophosphamide exposure increases ROS production, suggesting that biochemical and physiological disturbances may result from oxidative stress. 27 Quercetin decreased CP teratogenicity in our study. However, this property of quercetin was reported in other related studies. Quercetin (75 mg kg -1 ) had beneficial effect on serum lipid and glucose profile and minimized the monosodium glutamate related toxic effects, which was associated to its antioxidant properties. 28 Also, it has protected spinal cord against mechanism of inhibiting the activation of p38MAPK/iNOS signaling pathway and thus regulating secondary oxidative stress. 29 Quercetin with the dose of 66 mg kg -1 (low dose) and 333 mg kg -1 (high dose) throughout gestation, decreased placental oxidative stress and fetal skeletal malformation induced by methylnitrosourea. 30 Quercetin prevented renal tubular damage oxidative stress induced by chronic cadmium administration. 31 Hydroxyurea caused abnormal development of mouse embryos which is also reduced by quercetin. 32 Liang et al. reported that saturated fatty and lipid peroxidation related to fetal skeletal anomalies and quercetin (66 mg kg -1 supplemented diet) significantly improved their defects probably by its antioxidant effect on placenta. 33 This protective property of quercetin was demonstrated on alltrans-retinoic acid-induced teratogenicity when used at doses of 75 mg kg -1 and 200 mg kg -1 in rats on 8 th to10 th days of gestation. 20 In conclusion, the results of these studies are in consistent with the results of a recent study showing the ability of quercetin to reduce the damage caused by oxidative agents. Results of our study showed the effects of quercetin on elimination of CP induced teratogenicity for the first time. Taken together, 15 mg kg -1 CP on the 9 th day of pregnancy causes fetal malformations including cleft palate, exencephaly, spina bifida and skeletal abnormality in rat. Quercetin not only reduces skeletal abnormality but also protects weight and length abnormality of the fetus induced by CP. On the other hand, quercetin (200 mg kg -1 ) is more effective than quercetin (75 mg kg -1 ) in decreasing incidence CP-induced neural tube and skeletal defects in fetuses of rats. Therefore, antioxidant property of quercetin can protect the fetus against damage caused by CP.